Cyanide (CN), a prototype mitochondrial toxicant, produces progressive degeneration in select brain areas. This selective vulnerability to cyanide is characterized in vivo by apoptotic cell death in cortex and necrotic degeneration in substantia nigra. In primary cultured cells, cyanide produces a unique, cytotoxic response in which cortical cells (CX) undergo apoptosis and mesencephalic cells (MC) undergo necrosis, paralleling the regio-specific responses observed in animals. A rapid rise in cytosolic free Ca2+ and generation of reactive oxygen species are initiators of both modes of cell death. Preliminary studies show that two mitochondrial proteins (BNIP3 and UCP-2) are sensors of the initiation signals and function as co-regulators of cell death to determine which cell death pathway is executed. Proposed studies will characterize the role of BNIP3 and UCP-2 as sensors/regulators of cyanide-induced apoptosis and necrosis using primary cultured rat CX and MC cells as comparative models. To examine BNIP3 and UCP-2 as regulators of cyanide-induced cell death, changes in their expression will be produced, followed by monitoring subsequent changes in apoptotic and necrotic markers. Transient transfection with cDNA will be used to increase expression and RNA interference will produce knock down. For characterizing the link between BNIP3 and UCP-2 as functional co-regulators, cells will be co-transfected (cDNA or RNA interference) to concurrently increase or decrease expression of these proteins to determine if the mode of death can be modified temporally or switched. Upstream and downstream signaling cascades that initiate and execute BNIP3/UCP-2-mediated apoptosis and necrosis will be compared in the CX and MC expression models. Signals that activate BNIP3/UCP-2 will be characterized and then linked to recruitment of execution pathways. This will include determining how BNIP3/UCP-2 activation executes cell death by monitoring cellular redistribution/activity of apoptotic and necrotic signaling factors, including Bcl-2 proteins, cytochrome c, caspases, ATP levels and mitochondrial membrane pore transition. The long-term goal of this project is to identify mechanisms underlying cyanide-induced cell death and understand why brain areas are selectively vulnerable to cyanide. This study will provide valuable insight into neurotoxicant-induced neurodegeneration and fundamental information on regulation of cell death in the CNS.